电化学和紫外-可见吸收光谱法研究pH和金纳米粒子对细胞色素c的影响

制备了粒径均匀、平均粒子尺度为(4.7±0.6)nm,表面修饰3-巯基丙酸(MPA)的金纳米粒子(AuNPs).利用电化学和紫外-可见(UV-Vis)吸收光谱研究了pH和AuNPs对细胞色素c(Cytc)结构的影响.UV-Vis吸收光谱结果表明,pH为7.5-3.0时,Cytc和Cytc-AuNPs复合物的结构没有发生明显变化.当pH=2.0时,Cytc和Cytc-AuNPs复合物的Soret谱峰位置均发生明显移动,说明pH诱导其构象发生变化.循环伏安(CV)结果表明,表面修饰了MPA的AuNPs能促进Cytc和电极之间的电子传输,与修饰了柠檬酸根的AuNPs相比,其生物兼容性更好.pH的变化会引起CV中Cytc峰电流的改变和峰电位的移动.随着pH值的降低,Cytc电活性的量逐渐减小,并且pH诱导Cytc发生不可逆变性.AuNPs的引入使自由态的Cytc耐酸性增强,而使得吸附态的Cytc耐酸能力减弱.     

Surface plasmon resonance and electrochemistry characterization of layer-by-layer self-assembled DNA and Zr4+ thin films, and their interaction with cytochrome c

Through electrostatic layer-by-layer (LbL) assembly, negatively charged calf thymus double stranded DNA (CTds-DNA), and positively charged Zr⁴⁺ ions were alternately deposited on gold substrate modified with chemisorbed cysteamine. Thus-prepared three-dimensional DNA networks were characterized by surface plasmon resonance (SPR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and infrared reflection-absorption spectroscopy (IR-RAS). SPR spectroscopy indicates that the effective thickness of DNA monolayer in the (DNA/Zr⁴⁺)₁ bilayer was 1.5 ± 0.1 nm, which corresponds to the surface coverage of 79% of its full packed monolayer. At the same time, a linear increase of film thickness with increasing number of layers was also confirmed by SPR characterizations. The data of XPS and IR-RAS show that Zr⁴⁺ ions interact with both the phosphate groups and nitrogenous bases of DNA and load into the framework of DNA. Furthermore, the interactions between this composite film and heme protein cytochrome c (Cyt c) were investigated by SPR spectroscopy and electrochemistry. Compared with the adsorption of Cyt c on DNA monolayer, this composite multilayer film can obviously enhance the amount of immobilized Cyt c confirmed by SPR reflectivity-incident angle (R-θ) curves. Cyclic voltammetry (CV) indicates the Cyt c adsorbed on the composite film is electroactive, and the enhancement of peak current in CV indirectly verifies the increase of the amount of immobilized Cyt c.     

金纳米粒子比色测定细胞色素c构象变化

金纳米粒子(AuNPs)的颜色会随着细胞色素c(Cytc)构象变化而发生较大的变化,作者在改变原测定流程的基础上利用这种有规律的变化研究和测定了H+和L-半胱氨酸(L-Cys)对Cytc的构象变化.实验中分别加入pH=1-13的Cytc,可以使AuNPs显示青、蓝、紫、红等明显不同的颜色,从而可以利用AuNPs比色快速测定不同pH值的Cytc构象变化.在pH=7时,当附加L-Cys浓度从低浓度变化到高浓度时,AuNPs颜色逐渐从紫色变化到蓝色、青色,从而实现利用AuNPs比色测定由L-Cys所引起Cytc构象变化.圆二色(CD)光谱证实了pH=1-13和不同浓度L-Cys下的Cytc构象变化.借助紫外-可见吸收光谱和扫描电子显微镜(SEM)进一步明确了加入Cytc后AuNPs的不同聚集状态与其颜色变化的关系.     

Electrochemically controlled assembly and logic gates operations of gold nanoparticle arrays

The reversible assembly of β-cyclodextrin-functionalized gold NPs (β-CD Au NPs) is studied on mixed self-assembled monolayer (SAM), formed by coadsorption of redox-active ferrocenylalkylthiols and n-alkanethiols on gold surfaces. The surface coverage and spatial distribution of the β-CD Au NPs monolayer on the gold substrate are tuned by the self-assembled monolayer composition. The binding and release of β-CD Au NPs to and from the SAMs modified surface are followed by surface plasmon resonance (SPR) spectroscopy. The redox state of the tethered ferrocene in binary SAMs controls the formation of the supramolecular interaction between ferrocene moieties and β-CD-capped Au NPs. As a result, the potential-induced uptake and release of β-CD Au NPs to and from the surface is accomplished. The competitive binding of β-CD Au NPs with guest molecules in solution shifted the equilibrium of the complexation-decomplexation process involving the supramolecular interaction with the Fc-functionalized surface. The dual controlled assembly of β-CD Au NPs on the surface enabled to use two stimuli as inputs for logic gate activation; the coupling between the localized surface plasmon, associated with the Au NP, and the surface plasmon wave, associated with the thin metal surface, is implemented as readout signal for "AND" logic gate operations.     

Probing the Catalytic Activity of Reduced Graphene Oxide Decorated with Au Nanoparticles Triggered by Visible Light

Hybrid materials in which reduced graphene oxide (rGO) is decorated with Au nanoparticles (rGO–Au NPs) were obtained by the in situ reduction of GO and AuCl₄^?_(aq) by ascorbic acid. On laser excitation, rGO could be oxidized as a result of the surface plasmon resonance (SPR) excitation in the Au NPs, which generates activated O₂ through the transfer of SPR‐excited hot electrons to O₂ molecules adsorbed from air. The SPR‐mediated catalytic oxidation of p‐aminothiophenol (PATP) to p,p′‐dimercaptoazobenzene (DMAB) was then employed as a model reaction to probe the effect of rGO as a support for Au NPs on their SPR‐mediated catalytic activities. The increased conversion of PATP to DMAB relative to individual Au NPs indicated that charge‐transfer processes from rGO to Au took place and contributed to improved SPR‐mediated activity. Since the transfer of electrons from Au to adsorbed O₂ molecules is the crucial step for PATP oxidation, in addition to the SPR‐excited hot electrons of Au NPs, the transfer of electrons from rGO to Au contributed to increasing the electron density of Au above the Fermi level and thus the Au‐to‐O₂ charge‐transfer process.     

电化学研究聚乙二醇单甲醚接枝壳聚糖的pH敏感性及对细胞色素c的控制释放

采用电化学沉积法在Au电极上制备了聚乙二醇单甲醚接枝壳聚糖（mPEG-g-CS）电沉积膜及其与细胞色素c（Cyt c）复合膜。 循环伏安（CV）和交流阻抗（EIS）等电化学手段研究表明,mPEG-g-CS膜具有良好的pH敏感性,通过调节溶液的pH值,可以改变电化学探针[Fe(CN)6]3-/4-在电极表面的电子转移速率;Cyt c在mPEG-g-CS膜内可以实现直接电子转移,该复合膜电极对H2O2的响应快速而灵敏,说明mPEG-g-CS能较好的保持Cyt c的生物活性。 通过控制溶液的pH值,实现了Cyt c在mPEG-g-CS膜内的可逆释放和关闭。     

Folding control and unfolding free energy of yeast iso-1-cytochrome c bound to layered zirconium phosphate materials monitored by surface plasmon resonance

The free energy change (Delta G degrees ) for the unfolding of immobilized yeast iso-1-cytochrome c (Cyt c) at nanoassemblies was measured by surface plasmon resonance (SPR) spectroscopy. Data show that SPR is sensitive to protein conformational changes, and protein solid interface exerts a major influence on bound protein stability. First, Cyt c was self-assembled on the Au film via the single thiol of Cys-102. Then, crystalline sheets of layered alpha-Zr(O(3)POH)(2).H(2)O (alpha-ZrP) or Zr(O(3)PCH(2)CH(2)COOH)(2).xH(2)O (alpha-ZrCEP) were adsorbed to construct alpha-ZrP/Cyt c/Au or alpha-ZrCEP/Cyt c/Au nanoassemblies. The construction of each layer was monitored by SPR, in real time, and the assemblies were further characterized by atomic force microscopy and electrochemical studies. Thermodynamic stability of the protein nanoassembly was assessed by urea-induced unfolding. Surprisingly, unfolding is reversible in all cases studied here. Stability of Cyt c in alpha-ZrP/Cyt c/Au increased by approximately 4.3 kJ/mol when compared to the unfolding free energy of Cyt c/Au assembly. In contrast, the protein stability decreased by approximately 1.5 kJ/mol for alpha-ZrCEP/Cyt c/Au layer. Thus, OH-decorated surfaces stabilized the protein whereas COOH-decorated surfaces destabilized it. These data quantitate the role of specific functional groups of the inorganic layers in controlling bound protein stability.     

Adsorption of carboxylate-modified gold nanoparticles on an octadecylamine monolayer at the air/liquid interface

Gold nanoparticles (Au NPs) were prepared and surface-modified by mercaptosuccinic acid (MSA) to render a surface with carboxylic acid groups (MSA-Au). Octadecylamine (ODA) was used as a template monolayer to adsorb the Au NPs dispersed in the subphase. The effect of MSA concentration on the incorporation of Au NPs on the ODA monolayer and the relevant behavior of the mixed monolayer were studied using the pressure-area (pi-A) isotherm and transmission electron microscopy (TEM) observations. The experimental results showed that the adsorbed density of Au NPs is low without the surface modification by MSA. When MSA was added into the Au NP-containing subphase, the incorporation amount of Au NPs increased with increasing MSA concentration up to approximately 1 x 10-5 M for the particle density of 1.3 x 1011 particles/mL. With a further increase in the MSA concentration, the adsorbed particle density decreases due to competitive adsorption between the free MSA molecules and the MSA-Au NPs. It is inferred that free MSA molecules adsorb more easily than the MSA-Au NPs on the ODA monolayer. Therefore, an excess amount of MSA present in the subphase is detrimental to the incorporation of gold particles. The study on the monolayer behavior also shows that the pi-A isotherm of the ODA monolayer shifts right when small amounts of Au NPs or free MSA molecules are incorporated. However, when larger amounts of particles are adsorbed at the air/liquid interface, a left shift of the pi-A isotherm appears, probably due to the adsorption of ODA molecules onto the particle surface and the transferring of the particles from beneath the ODA monolayer to the air/water interface. According to the present method, it is possible to prepare uniform particulate films of controlled densities by controlling the particle concentration in the subphase, the MSA concentration, and the surface pressure of a mixed monolayer.     

Enzyme-free surface plasmon resonance aptasensor for amplified detection of adenosine via target-triggering strand displacement cycle and Au nanoparticles

Herein, we combine the advantage of aptamer technique with the amplifying effect of an enzyme-free signal-amplification and Au nanoparticles (NPs) to design a sensitive surface plasmon resonance (SPR) aptasensor for detecting small molecules. This detection system consists of aptamer, detection probe (c-DNA1) partially hybridizing to the aptamer strand, Au NPs-linked hairpin DNA (Au-H-DNA1), and thiolated hairpin DNA (H-DNA2) previously immobilized on SPR gold chip. In the absence of target, the H-DNA1 possessing hairpin structure cannot hybridize with H-DNA2 and thereby Au NPs will not be captured on the SPR gold chip surface. Upon addition of target, the detection probe c-DNA1 is forced to dissociate from the c-DNA1/aptamer duplex by the specific recognition of the target to its aptamer. The released c-DNA1 hybridizes with Au-H-DNA1 and opens the hairpin structure, which accelerate the hybridization between Au-H-DNA1 and H-DNA2, leading to the displacement of the c-DNA1 through a branch migration process. The released c-DNA1 then hybridizes with another Au-H-DNA1 probe, and the cycle starts anew, resulting in the continuous immobilization of Au-H-DNA1 probes on the SPR chip, generating a significant change of SPR signal due to the electronic coupling interaction between the localized surface plasma of the Au NPs and the surface plasma wave. With the use of adenosine as a proof-of-principle analyte, this sensing platform can detect adenosine specifically with a detection limit as low as 0.21 pM, providing a simple, sensitive and selective protocol for small target molecules detection.     

Au NPs-aptamer conjugates as a powerful competitive reagent for ultrasensitive detection of small molecules by surface plasmon resonance spectroscopy

Features of Au NPs-aptamer conjugates as a powerful competitive reagent to substitute antibody in enhancing surface plasmon resonance spectroscopy (SPR) signal for the detection of small molecule are explored for the first time. In order to evaluate the sensing ability of Au NPs-aptamer conjugates as a competitive reagent, a novel SPR sensor based on indirect competitive inhibition assay (ICIA) for the detection of adenosine is constructed by employing the competitive reaction between antiadenosine aptamer with adenosine and antiadenosine aptamer with its partial complementary ss-DNA. The partial complementary ss-DNA of antiadenosine aptamer is firstly immobilized on SPR gold film as sensing surface. When the Au NPs-antiadenosine aptamer conjugates solution is added to SPR cell in the absence of adenosine, Au NPs-antiadenosine aptamer conjugates is adsorbed to SPR sensor by the DNA hybridization reaction, and results in a large change of SPR signal. However, the change of SPR signal is decreased when the mixing solution of adenosine with Au NPs-antiadenosine aptamer conjugates is added. This is because adenosine reacts with antiadenosine aptamer in Au NPs-antiadenosine aptamer conjugates and changes its structure from ss-DNA to tertiary structure, which cannot hybridize with its partial complementary ss-DNA immobilized on SPR gold surface. Based on this principle, a SPR sensor for indirect detection of adenosine can be developed. The experimental results confirm that the SPR sensor possesses a good sensitivity and a high selectivity for adenosine, which indirectly confirms that Au NPs-aptamer conjugates is a powerful competitive reagent. More significantly, it can be used to develop other SPR sensors based on ICIA to detect different targets by changing the corresponding type of aptamer in Au NPs-aptamer conjugates.     

Reversible immobilization and direct electron transfer of cytochrome c on a pH-sensitive polymer interface

A pH-sensitive polymer interface has been used as a matrix for reversible immobilization of cytochrome c (Cyt c) on an Au surface through a dip-coating process. The pH-sensitive behavior of the polymer brush interface has been demonstrated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements. The reversible immobilization and electron-transfer properties of Cyt c have been investigated by in situ UV/Vis spectrophotometry and CV. The results have shown that the poly(acrylic acid) (PAA) brush acted as an excellent adsorption matrix and a good accelerant for the direct electron transfer of Cyt c, which gave redox peaks with a formal potential of 40 mV versus Ag/AgCl in pH 7.6 phosphate buffer solution. The average surface coverage of Cyt c on the PAA film was about 1.7 x 10(-10) mol cm(-2), indicating a multilayer of Cyt c. The electron-transfer rate constant was calculated to be around 0.19 s(-1) according to the CV experiments. The interface was subjected to in situ attenuated total internal reflection Fourier-transform infrared (ATR-FTIR) spectroscopic analysis, in order to further confirm the immobilization of Cyt c on the surface. This polymer-protein system may have potential applications in the design of biosensors, protein separation, interfacial engineering, biomimetics, and so on.     

AuAg bimetallic nanoparticles film fabricated based on H2O2-mediated silver reduction and its application

A method to fabricate AuAg bimetallic nanoparticles film by H₂O₂-mediated reduction of silver was reported. Gold nanoparticles (Au NPs) were first adsorbed onto the surface of a self-assembled 2-aminoethanethiol monolayer-modified gold film or 3-aminopropyltriethoxysilane (APTES) monolayer-modified quartz slide. Upon further treatment of this modified film with the solution containing silver nitrate (AgNO₃) and H₂O₂, silver was deposited on the surface of Au NPs. The size of the AuAg bimetallic particles could be readily tuned by manipulating the concentration of H₂O₂. Surface plasmon resonance (SPR) was used to investigate the process, the deposition of silver on Au NPs modified gold film resulted in an obvious decrease of depth in the SPR reflectance intensity and minimum angle curves (SPR R-θ curves), which may be utilized for the quantitative SPR detection of the analyte, H₂O₂. Combination of the biocatalytic reaction that could yield H₂O₂ by using the enzyme, glucose oxidase, with the deposition of silver may enable the design of a glucose biosensor by SPR technique. Furthermore, we evaluated the AuAg bimetallic nanoparticles film for their ability to be an effective substrate for surface-enhanced Raman scattering (SERS).     

纳米金三明治结构调制细胞色素c电子传递特性的分子机理研究

以细胞色素c(Cyt c)为模型蛋白,采用表面增强红外吸收光谱监测了三明治结构所吸附的纳米金对氧化还原诱导的Cyt c表面增强红外差谱的改变.研究表明,在单层Cyt c分子表面组装纳米金,使得血红素的红外差谱特征峰明显增强,这归因于纳米金和血红素之间的电子传递.纳米金与Cyt c氧化还原活性中心血红素的相互作用加速了蛋白质的电子传递.这为实现并优化表面吸附蛋白质的直接电化学提供了一种新技术.     

Assessment of Defects and Collapsed Sites in Self-Assembled Alkanethiol Monolayer on Gold by Cyclic Voltammetry and A.C.Impedance

Thespontaneousself-assemblytechniqueprovidesaconvenientandpowerfultooltopreparecompactmonolayerswithwell-definedcomposition,structureandthicknessl.However,theexistenceofstrUcturaldefectsanddisorderinself-assemblymayinfluenceitspropertiesinmanyfields.Soitisincreasinglyclearthatthecharacterizationofdefectsisanimportantissuewhichneedstobeaddressedinmanyapplications.Thepurposeofthisworkistostudythedefectsand"collapsed"sitesinAulthiolmonolayerbycyclicvoltanunetryandelectrochemicalimpedancespectros…     

Amplified surface plasmon resonance based DNA biosensors, aptasensors, and Hg2+ sensors using hemin/G-quadruplexes and Au nanoparticles

Thiolated nucleic acid hairpin nanostructures that include in their stem region a "caged" G-quadruplex sequence, and in their single-stranded loop region oligonucleotide recognition sequences for DNA, adenosine monophosphate (AMP), or Hg(2+) ions were linked to bare Au surfaces or to Au nanoparticles (NPs) linked to Au surfaces. The opening of the hairpin nanostructures associated with the bare Au surface by the complementary target DNA, AMP substrate, or Hg(2+) ions, in the presence of hemin, led to the self-assembly of hemin/G-quadruplexes on the surface. The resulting dielectric changes on the surface exhibited shifts in the surface plasmon resonance (SPR) spectra, thus providing a readout signal for the recognition events. A similar opening of the hairpin nanostructures, immobilized on the Au NPs associated with the Au surface, by the DNA, AMP, or Hg(2+) led to an ultrasensitive SPR-amplified detection of the respective analytes. The amplification originated from the coupling between the localized surface plasmon associated with the NPs and the surface plasmon wave, an effect that cooperatively amplifies the SPR shifts that result from the formation of the hemin/G-quadruplexes. The different sensing platforms reveal impressive sensitivities and selectivities toward the target analytes.     

Electrochemical study of self-assembled monolayer adsorption

The electrochemical behaviour of self-assembled monolayer (SAM) of aliphatic hexadecanethiol was studied by cyclic voltammetry (CV), elimination voltammetry with linear scan (EVLS) and crystal quartz microbalance (QCM). SAMs were electrochemically created on gold-coated QCM crystal through the sulphur in 1-hexadecanethiol molecule head group. The effect of thiol concentration and potential scan rate on the SAM formation was studied. Formation of SAM was confirmed by CV and QCM. EVLS results revealed the kinetically controlled process followed with electrode reaction in adsorbed state characteristic for SAM formation at lower concentration. The electrode reaction of a totally adsorbed electroactive species was indicated by means of a peak-counter peak signal at higher thiol concentration.     

Extraordinary enhancement of Raman scattering from pyridine on single crystal Au and Pt electrodes by shell-isolated Au nanoparticles

We used shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) to systematically study the adsorption of pyridine on low-index Au(hkl) and Pt(hkl) single crystal electrodes. Our gold-core silica-shell nanoparticles (Au@SiO(2) NPs) boost the intensity of Raman scattering from molecules adsorbed on atomically flat surfaces. The average enhancement factor reaches 10(6) for Au(110) and 10(5) for Pt(110), which is comparable to or even greater than that obtained for bare gold NPs (a widely adopted SERS substrate). 3D-FDTD simulations reveal that this large enhancement is due to the transfer of the "hotspots" from NP-NP gaps to NP-surface gaps. We also found that the SHINERS intensity strongly depends on the surface crystallographic orientation, with differences up to a factor of 30. Periodic DFT calculations and theoretical analysis of dielectric functions indicate that this facet-dependence is predominantly governed by the dielectric property of the surface. The results presented in this work may open up new approaches for the characterization of adsorbates and reaction pathways on a wide range of smooth surfaces.     

Fabrication of electroactive layer-by-layer films of myoglobin with gold nanoparticles of different sizes

Alternate adsorption of oppositely charged myoglobin (Mb) and gold nanoparticles with different sizes were used to assemble {Au/Mb}n layer-by-layer films on solid surfaces by electrostatic interaction between them. The direct electrochemistry of Mb was realized in {Au/Mb}n films at pyrolytic graphite (PG) electrodes, showing a pair of well-defined, nearly reversible cyclic voltammetry (CV) peaks for the Mb heme FeIII/FeII redox couple. Quartz crystal microbalance (QCM), electrochemical impedance spectroscopy (EIS), and CV were used to monitor or confirm the growth of the films. Compared with other Mb layer-by-layer films with nonconductive nanoparticles or polyions, {Au/Mb}n films showed much improved properties, such as smaller electron-transfer resistance (Rct) measured by EIS with Fe(CN)3-/4- redox probe, higher maximum surface concentration of electroactive Mb (Gamma*max), and better electrocatalytic activity toward reduction of O2 and H2O2, mainly because of the good conductivity of Au nanoparticles. Because of the high biocompatibility of Au nanoparticles, adsorbed Mb in the films retained its near native structure and biocatalytic activity. The size effect of Au nanoparticles on the electrochemical and electrocatalytic activity of Mb in {Au/Mb}n films was investigated, demonstrating that the {Au/Mb}n films assembled with smaller-sized Au nanoparticles have smaller Rct, higher Gamma*max, and better biocatalytic reactivity than those with larger size.     

Changes in adsorbed fibrinogen upon conversion to fibrin

The conversion of adsorbed fibrinogen to fibrin in the presence of the enzyme thrombin was studied using surface plasmon resonance (SPR), a quartz crystal microbalance (QCM), sum frequency generation (SFG), atomic force microscopy (AFM), and an elutability assay. Exposure of adsorbed fibrinogen to thrombin resulted in a mass loss at the surface consistent with fibrinopeptide release and conversion to fibrin. Changes in hydration upon conversion of adsorbed fibrinogen to fibrin were determined from comparisons of acoustic (QCM) and optical (SPR) mass adsorption data. Conversion to fibrin also resulted in the adsorbed layer becoming more strongly bound to the surface and more compact. The elutability of adsorbed fibrinogen by Triton X-100, studied with SPR, decreased from 90 +/- 5 to 6 +/- 2% after conversion to fibrin. The height of the adsorbed monolayer, as determined by AFM, decreased from 5.5 +/- 2.2 to 1.7 +/- 0.8 nm. We conclude that thrombin-catalyzed fibrinopeptide release triggers significant changes in fibrinogen conformation beyond peptide cleavage.     

Building a novel vitronectin assay by immobilization of integrin on calixarene monolayer

Membrane proteins possess significant hydrophobic domains and are likely to deplete their native activity immobilized on the solid surface relative to those occurring in a membrane environment. To investigate an efficient immobilization method, calix[4]crown-ether monolayer as an artificial protein linker system was constructed on the gold surface and characterized by Fourier transform infrared reflection absorption spectroscopy (FTIR-RAS), atomic force microscopy (AFM) and cyclic voltammetry (CV). Integrin alpha(v)beta3 was functionally immobilized onto the monolayer and the integrin-vitronectin interaction was investigated by surface plasmon resonance (SPR). It was found that calix[4]crown-ether was assembled as a monolayer on the gold surface. Orientation and accessibility of integrin alpha(v)beta3 was assessed by sensitive binding of its natural ligand, vitronectin at pg mL(-1) level. Moreover, surface coverage of integrin layer and thickness calculated through SPR curve simulation verified that integrin layer was a monolayer in activated form. In combination with the SPR method, this calix[4]crown monolayer provided a reliable and simple experimental platform for the investigation of isolated membrane proteins under experimental conditions resembling those of their native properties.